Component of von Hippel-Lindau tumor suppressor complex and SCF ubiquitin ligase

ABSTRACT

Rbx1, an evolutionarily conserved Cullin-interacting RING-H2 finger protein, has been discovered. Mammalian Rbx1 has been identified as a component of the CUL2-containing VHL complex. An Rbx1 homolog from  S. cerevisiae  has also been identified as a subunit and activator of the Cdc53-containing SCF Cdc4  ubiquitin ligase required for ubiquitination of the cdk inhibitor Sic1 and for the G1/S cell cycle transition in yeast, providing a link between the multiprotein VHL tumor suppressor complex and cellular ubiquitination. The Rbx1 protein acts as a cellular marker useful (1) in detecting a possible predisposition to certain carcinomas, (2) as a molecular target for treating those carcinomas therapeutically. (3) as a target for inhibition by drugs that manipulate the growth of cells, and (4) as a research tool to better understand the various complex mechanisms of cell ubiquitination, binding of certain activator proteins, fibronectin deposition and other aspects of the cellular division process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and all rights of priority toU.S. Provisional Application Ser. No. 60/121,787, filed Feb. 26, 1999,which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a bio-affecting and body treating compositionand to a biological diagnostic agent.

BACKGROUND OF THE INVENTION

The von Hippel-Lindau (VHL) tumor suppressor gene on chromosome 3p25.5is mutated in the majority of sporadic clear cell renal carcinomas andin VHL disease, an autosomal dominant familial cancer syndrome thatpredisposes affected individuals to a variety of tumors including clearcell renal carcinomas, cerebellar hemangioblastomas and hemangiomas,retinal angiomata, and pheochromocytomas. (Linehan, et al. 1995.“Identification of the von Hippel-Lindau (VHL) gene. Its role in renalcancer,” JAMA273:564-570; Gnarra, et al. 1996. “Molecular cloning of thevon Hippel-Lindau tumor suppressor gene and its role in renalcarcinoma,” Biochim Biophys Acta 1242;201-210; and Kaelin, W. G. and E.R. Maher. 1998. “The VHL tumor-suppressor gene paradigm,” Trends Genet14:423-426). The VHL protein is expressed in most tissues and cell typesand appears to perform multiple functions, including general repressionof hypoxia-inducible genes (Iliopoulos, et al. 1996. “Negativeregulation of hypoxia-inducible genes by the von Hippel-Lindau protein,”Proc Natl Acad Sci USA 93:10595-10599; Gnarra, et al. 1996.Post-transcriptional regulation of vascular endothelial growth factormRNA by the product of the VHL tumor suppressor gene,” Proc Natl AcadSci USA 93:10589-10594; and Siemeister, et al. 1996. “Reversion ofderegulated expression of vascular endothelial growth factor in humanrenal carcinoma cells by von Hippel-Lindau tumor suppressor protein,”Cancer Res 56:2299-2301); regulation of p27 protein stability (Pause, etal. 1998. “The von Hippel-Lindau tumor suppressor gene is required forcell cycle exit upon serum withdrawal,” Proc Natl Acad Sci USA95:993-998; and Kim, et al. 1999. “Recombinant adenovirus expressing VonHippel-Lindau-mediated cell cycle arrest is associated with theinduction of cyclin-dependent kinase inhibitor p27Kipl,” BBRC253:672-677); and regulation of the assembly of extracellularfibronectin matrix (Ohh, et al. 1998. “The von Hippel-Lindau tumorsuppressor protein is required for proper assembly of an extracellularfibronectin matrix,” Mol Cell 1:959-968).

In all cell types examined, the VHL protein is found in a multiproteincomplex that includes the ubiquitin-like Elongin B protein, and ElonginC and the cullin CUL2, which share sequence similarity with the Skp1 andCdc53 components of the Skp1-Cdc53p-F-box protein (SCF) ubiquitin ligasecomplex, respectively. (Kibel, et al. 1995. “Binding of the vonHippel-Lindau tumor suppressor protein to Elongin B and C,” Science269:1444-1446; Duan, et al. 1995. “Inhibition of transcriptionelongation by the VHL tumor suppressor protein,” Science 269:1402-1406;Pause, et al. 1997. “The von Hippel-Lindau tumor-suppressor gene productforms a stable complex with human CUL-2, a member of the Cdc53 family ofproteins,” Proc Natl Acad Sci USA 94:2156-2161; and Lonergan, et al.1998. “Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindautumor suppressor protein requires binding to complexes containingElongins B/C and Cul2,” Mol Cell Biol 18:732-741). Elongins B and C forma stable subcomplex that interacts with a short BC-box motif in the VHLprotein and bridges its interaction with CUL2. (Pause, et al. 1997. ProcNatl Acad Sci USA 94:2156-2161; Lonergan, et al. 1998. Mol Cell Biol18:732-741). A large fraction of VHL mutations found in sporadic clearcell renal carcinomas and in VHL kindreds result in mutation or deletionof the BC-box, disruption of the VHL complex, and deregulation ofhypoxia-inducible gene expression, p27 protein stability, and bironectinmatrix assembly. (Gnarra, et al. 1996. Biochim Biophys Acta1242:201-210; Pause, et al. 1997. Proc Natl Acad Sci USA 94:2156-2161;Lonergan, et al. 1998. Mol Cell Biol 18:732-741; Ohh, et al. 1998. MolCell 1:959-968; and Kaelin, W. G. and E. R. Maher. 1998. Trends Genet14:423-426).

Despite information currently available regarding the multiproteincomplex that includes the VHL protein, Elongin B, Elongin C, and CUL2,not all components of this complex and the binding relationship of thesecomponents to one another have been elucidated. There is a continuingneed to characterize yet unknown components of the complex and theirinteraction with other components to further the understanding of thecomplex and its relationship to disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C describe the co-purification of the VHL complex with Rbx1from rat liver cytosol.

FIG. 1A is a schematic showing the various steps used in thepurification of the VHL complex, wherein P-cell=phosphocellulose P11.

FIG. 1B is a chromatography of Rbx1 with the VHL complex, whereinVHL=von Hippel-Lindau protein; CUL2=CUL2 protein or cullin; EloB−ElonginB; EloC=Elongin C. Aliquots of column fractions from the MonoQ columnwere subjected to 12% SDS-polyacrylamide gel electrophoresis, andproteins were detected by silver staining.

FIG. 1C depicts a 5-20% SDS-polyacrylamide gel of sample used forpeptide sequencing.

FIG. 2 is a chart showing the alignment of predicted Rbx1 proteinsequences from human (SEQ ID NO:1), mouse (SEQ ID NO:1), Drosophilamelanogaster (SEQ ID NO:6), Caenorhabditis elegans (SEQ ID NO:7), andSaccharomyces cerevisiae (SEQ ID NO:2) with APC11 from S. cerevisiae(SEQ ID NOS:8 and 9), wherein DROS=Drosophila melanogaste;ELEGANS=Caenorhabditis elegans; and YEAST=Saccharomyces cerevisiae. Thealignment was generated using the MACAW program. (Schuler, et al. 1991.“A workbench for multiple alignment construction and analysis,”Proteins; Struct Funct Genet 9:180-190). Dark shading indicatespositions of identity between Rbx1 proteins from different species andpositions of identity between the Rbx1 and APC11 proteins. Grey shadingindicates positions of similaiy.

FIGS. 3A-3D depict the reconstitution of Rbx1-containing complexes.

FIG. 3 a demonstrates Rbx1 forming complexes with VHL and CUL2 in thepresence of Elongin BC. Lysates from Sf21 cells expressing the indicatedviruses were immunoprecipitated with anti-FLAG or anti-MYC antibodies,and immunoprecipitated proteins were detected by immunoblotting.

FIG. 3B demonstrates independent association of Rbx1 with Elongin BC,and VHL. Lysates from Sf21 cells expressing the indicated viruses wereimmunoprecipitated with anti-HPC4, anti-FLAG, or anti-MYC antibodies,and immunoprecipitated proteins were detected by inmnunoblotting.

FIG. 3C demonstrates in vitro binding of recombinant Rbx1, VHL, andElongin BC. Proteins expressed in and purified from E. coli were mixedtogether in the combinations indicated, renatured by dilution anddialysis, and immunoprecipitated with anti-HPC4. Immunoprecipitatedproteins were detected by immunoblotting with the indicated antibodies.

FIG. 3D demonstrates independent association of Rbx1 with Cul1, Cul2,and Cdc53. Lysates from Sf21 cells expressing the indicated viruses wereimmunoprecipitated with anti-MYC antibodies, and immunoprecipitatedproteins were detected by immunoblotting.

FIGS. 4A-4C depict various activities associated with the presence orabsence of Rbx1.

FIG. 4A demonstrates that binding of Rbx1 protein to endogenous yeastCdc53 correlates with function. The upper panel shows phenotypes ofrbx1Δ cells expressing wild type or mutant mammalian Rbx1 (mRbx1)protein. As shown in the lower panel, lysates from cells expressing wildtype and mutant manmnalian MYC-Rbx1 proteins in either the rbx1 deletionstrain (deleted) or in the parental strain MCY453 (wild type) weresubjected to immunoprecipitation with anti-MYC antibodies.Immunoprecipitated proteins were detected by immunoblotting withanti-MYC or anti-Cdc53 antibodies.

FIG. 4B demonstrates that Sic1 protein accumulates in Rbx1-depletedcells. Rbx1Δ/pGAL-mrbx1 (M4) cells were grown to an OD600 of 1 ingalactose-containing medium and then shifted into glucose medium. Cellswere harvested after 8 hours of growth in glucose, and cell lysates wereanalyzed by immunoblotting with anti-MYC and two different anti-Sic1antibodies.

FIG. 4C demonstrates morphological changes associated withRbx1-depletion. Rbx1Δ/pGAL-mrbx1 (M4) cells were grown in galactose(gal) or for 8 hours after glucose shift (glu) prior to fixation andstaining. Nuclear morphology was visualized by DAPI(4,6-diamidino-2-phenylindole) staining. (Panels A and C. DIC; panels Band D, DAPI).

FIG. 5 shows a diagram of a SCF ubiquitin ligase complex and a VHLubiquitin ligase complex and illustrates the ubiquitin ligases of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “Ring box protein” and “Rbx1” as used herein refers toproteins that are components of the von Hippel-Lindau tumor suppressorcomplex. The terms refer to proteins obtained from any eukaryoticspecies, particularly mammalian species such as bovine, porcine, murine,equine, and human, and from any source whether natural, synthetic,semi-synthetic, or recombinant. The terms encompass polypeptides orproteins having less than the complete amino acid sequence andbiologically active variants and gene products.

The term “naturally occurring” as used herein means an endogenous orexogenous protein isolated and purified from animal tissue or cells.

The term “isolated and purified” as used herein means a protein that isessentially free of association with other proteins or polypeptides,e.g., as a naturally occurring protein that has been separated fromcellular and other contaminants by the use of antibodies or othermethods or as a purification product of a recombinant host cell culture.

The term “biologically active” as used herein means a protein havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule.

The term “nucleotide sequence” as used herein means a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct that has been derived from DNA or RNAisolated at least once in substantially pure form (ie., free ofcontaminating endogenous materials) and in a quantity or concentrationenabling identification, manipulation, and recovery of its componentnucleotide sequences by standard biochemical methods. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal non-translated sequences, or introns that are typicallypresent in eulkaryotic genes. Sequences of non-translated DNA may bepresent 5′ or 3′ from an open reading frame where the same do notinterfere with manipulation or expression of the coding region.

The term “nucleic acid molecule” as used herein means RNA or DNA,including cDNA, single or double stranded, and linear or covalentlyclosed molecules. A nucleic acid molecule may also be genornic DNAcorresponding to the entire gene or a substantial portion thereof or tofragments and derivatives thereof. The nucleotide sequence maycorrespond to the naturally occurring nucleotide sequence or may containsingle or multiple nucleotide substitutions, deletions and/or additionsincluding fragments thereof. All such variations in the nucleic acidmolecule retain the ability to encode a biologically active protein whenexpressed in the appropriate host or a biologically active fragmentthereof The nucleic acid molecule of the present invention may comprisesolely the nucleotide sequence encoding a protein or may be part of alarger nucleic acid molecule that comprises the gene for ]extends to thegene for the protein. The non-protein encoding sequences in a largernucleic acid molecule may include vector, promoter, terminator,enhancer, replication, signal sequences, or non-coding regions of thegene.

The term “variant” as used herein means a polypeptide substantiallyhomologous to a naturally occurring protein but which has an amino acidsequence different from the naturally occurring protein (human, bovine,ovine, porcine, murine, equine, or other eukaryotic species) because ofone or more deletions, insertions, derivations, or substitutions. Thevariant amino acid sequence preferably is at least 40% identical to anaturally occurring amino acid sequence but is most preferably at least70% identical. Variants may comprise conservatively substitutedsequences wherein a given amino acid residue is replaced by a residuehaving similar physiochemical characteristics. Conservativesubstitutions are well known in the art and include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Conventionalprocedures and methods can be used for making and using such variants.Other such conservative substitutions such as substitutions of entireregions having similar hydrophobicity characteristics are well known.Naturally occurring variants are also encompassed by the presentinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of theprotein that leave the protein biologically active and capable ofperforming its biological function. Alternate splicing of mRNA may yielda truncated but biologically active protein. Variations attributable toproteolysis include differences in the N- or C-termini upon expressionin different types of host cells due to proteolytic removal of one ormore terminal amino acids from the protein.

The term “substantially the same” as used herein means nucleic acid oramino acid sequences having sequence variations that do not materiallyaffect the nature of the protein, ie., the structure and/or biologicalactivity of the protein. With particular reference to nucleic acidsequences, the term “substantially the same” is intended to refer to thecoding region and to conserved sequences governing expression and refersprimarily to degenerate codons encoding the same amino acid or alternatecodons encoding conservative substitute amino acids in the encodedpolypeptide. With reference to amino acid sequences, the term“substantially the same” refers generally to conservative substitutionsand/or variations in regions of the polypeptide not involved indetermination of structure or function.

The term “percent identity” as used herein means comparisons among aminoacid sequences as defined in the UWGCG sequence analysis programavailable from the University of Wisconsin. (Devereaux et al., Nucl.Acids Res. 12: 387-397 (1984)).

This invention is not limited to the particular methodology, protocols,cell lines, vectors, and reagents described because these may vary.Further, the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to limit the scope ofthe present invention. As used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise, e.g. reference to “a host cell”includes a plurality of such host cells.

Because of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding Rbx1 or other Ring box proteins referred to hereinmay be produced. Some of these sequences will be highly homologous andsome will be minimally homologous to the nucleotide sequences of anyknown and naturally occurring gene. The present invention contemplateseach and every possible variation of nucleotide sequence that could bemade by selecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurring Rbx1and all such variations are to be considered as being specificallydisclosed.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein byreference to the extent allowed by law for the purpose of describing anddisclosing the proteins, enzymes, vectors, host cells, and methodologiesreported therein that might be used with the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

The Invention

The present invention provides isolated and purified biologically activecullin-interacting RING-H2 finger proteins (“Ring box proteins”) thatare a component of the von Hippel-Lindau tumor suppressor complex and ofSCF ubiquitin ligase complexes, nucleic acid molecules that encode Ringbox proteins, expression vectors having DNA that encode Ring boxproteins, host cells that have been transfected or tnansformed withexpression vectors having DNA that encode Ring box proteins, methods forproducing recombinant Ring box proteins by culturing host cells thathave been transfected or transformed with expression vectors having DNAthat encode Ring box proteins, isolated and purified recombinant Ringbox proteins, complexes and methods that use Ring box proteins to screenfor therapeutic agents, methods for diagnosing the predisposition of apatient to certain carcinomas, methods for treating any of severalenumerated carcinomas or augmenting metabolically deficient systems inhumans and other animals, and methods for evaluating the effectivenessof a therapeutic treatment for Ring box associated carcinomas. Inparticular, the present invention provides a new Ring box proteindesignated Rbx1.

Isolation and Purification of Rbx1

The endogenous VHL complex from rat liver was first purified by theprocedures given below and outlined in FIG. 1A. The VHL complex waspurified from a post-nuclear supernatant prepared from the livers of 360male Sprague-Dawley rats and fractionated by (NH₄)₂ SO₄ precipitation asdescribed previously. (Conaway, et al. 1996. “Purification of RNApolymerase II general transcription factors from rat liver,” MethEnzymol 273:194-207). Fractions containing the VHL complex wereidentified by Western blotting using IG32, a monoclonal against the VHLprotein. (Kibel, et al. 1995. “Binding of the von Hippel-Lindau tumorsuppressor protein to Elongin B and C,” Science 269:1444-1446). The 0 to38% (NH₄)₂ SO₄ fraction was resuspended in Buffer A [20 mM Hepes-NaOH(pH 7.9), 0.5 mM EDTA, 1 mM DTT, 10% (v/v) glycerol] containing 0.5 MMPMSF, 10 μg/ml antipain, and 10 μg/ml leupeptin and was brought to aconductivity equivalent to that of Buffer A containing 100 mM KCl by a 3hour dialysis against Buffer A containing 0.5 mM PMSF and dilution withthe same buffer. The dialysate was centrifuged for 30 min at 28,000×g,and the supernatant was gently mixed for˜45 minutes with 800 ml ofphosphocellulose (P11, Whatnan) preequlibrated in Buffer A containing100 Mm M KCl and 0.5 mM PMSF. The slurry was then filtered at 500 ml/hrin a 10.5-cm diameter column. The phosphocellulose flow through fractionwas gently mixed for˜45 minutes with 250 ml Toyopearl DEAE-650 M(TosoHaas) pre-equilibrated in Buffer C [40 mM Tris-HCl (pH 7.9), 0.5 mMEDTA, 1 M DTT, and 10% (v/v) glycerol] containing 80 mM KCl. The slurrywas filtered at 150 ml/hr in a 5.0-cm diameter column and then washed atthe same flow rate with Buffer C containing 80 mM KCl. The column waseluted stepwise at 250 ml/hr with Buffer A containing 220 mM KCl, and 50ml fractions were collected. Fractions containing the VHL protein wereconcentrated by 0.3 mg/ml (NH₄)₂ SO₄ precipitation, resuspended in 10 mlof Buffer A containing 10 μg/ml antipain and 10 μg/ml leupeptin, anddialyzed against Buffer A containing 300 mM KCl to a conductivityequivalent to that of Buffer A containing 500 mM (NH₄)₂SO₄. Followingcentrifugation for 15 min at 12,000×g, the resulting supernatant wasapplied at 20 ml/hr to a 500 ml, 2.6-cm diameter Ultrogel AcA 34 gelfiltration column (IBF Biotechnics) pre-equilibrated in Buffer Acontaining 400 mM KCl. The column was eluted at 20 ml/hr, and 10 mlfractions were collected. Fractions containing the VHL protein, whicheluted with an apparent native molecular mass between 330 kDa and 200kDa, were diluted with an equal volume of Buffer E [40 mM Hepes-NaOH (pH7.9), 0.1 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol] containing 2.0 M(NH₄)₂ SO₄. Following centrifugation for 20 min at 60,000×g, theresulting supernatant was applied to a Spherogel TSK phenyl 5-PW column(21.5-×150-mm, Beckman Instruments) pre-equilibrated in Buffer Econtaining 1.0 M (NH₄)₂SO₄. The column was eluted at 5 ml/min with a 250ml linear-gradient from 1.0 M (NH₄)₂ SO₄ in Buffer E to Buffer E, and 5ml fractions were collected. Fractions containing the VHL protein, whicheluted between 170 mM and 80 mM (NH₄)₂SO₄, were pooled and dialyzedagainst Buffer C to a conductivity equivalent to that of Buffer Ccontaining 60 mM KCl. Following centrifugation for 20 min at 60,000×g,the resulting supernatant was applied to a BioGel SEC DEAE 5-PW column(7.5-×75-mm, Bio-Rad) pre-equilibrated in Buffer C containing 60 mM KCl.The column was eluted at 0.8 mu/min with a 40 ml linear gradient from 60mM to 250 mM KCl in Buffer C, and 0.7 ml fractions were collected.Fractions containing the VHL protein, which eluted between 120 mM and140 mM KCl, were pooled and brought to 5 mM potassium phosphate.Following centrifilgation for 20 min at 60,000×g, the resultingsupernatant was applied to a crystalline hydroxylapatite Bio-Scale CHT-Icolumn (7-×52-mm, Bio-Rad) pre-equilibrated in Buffer P [5 mM potassiumphosphate (pH 7.6), 0.1 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol]. Thecolumn was eluted at 0.6 ml/min with a 24 ml linear gradient from 50 mMto 400 mM potassium phosphate (pH 7.6) in Buffer P, and 0.3 ml fractionswere collected. Fractions containing the VHL protein, which elutedbetween 50 mM and 80 mM potassium phosphate, were pooled and dilutedwith Buffer C containing 50 mM KCl to a conductivity equivalent to thatof Buffer C in 80 mM KCl. Following centrifugation for 20 min at60,000×g, the resulting supernatant was applied to a MonoQ column(5-×50-mm, Phanmacia) preequilibrated in Buffer C containing 80 mM KCl.The column was eluted at 0.4 ml/min with a 12 ml linear gradient from 80mM to 300 mM KCl in Buffer C, and 0.2 ml fractions were collected.Fractions containing the VHL protein eluted between 180 mM and 200 mMKCl.

As shown in FIGS. 1B and 1C, greater than 90% of the detectable VHLprotein in liver homogenates copurified with CUL2, Elongin B, Elongin C,and a small polypeptide with an apparent molecular mass of −16 kDa Theidentities of the VHL, CTL2, Elongin B, and Elongin C potypeptides wereconfirmed by Western blotting and/or peptide sequencing. On-line iontrap HPLC/MS/MS peptide sequencing using the method described below(Eng, et al. 1994. J Am Soc Mass Spectrom 5:976 and Chittum et al. 1998.Biochemisty 37:10866) of the −16 kDa protein excised from the SDSpolyacrylamide gel shown in FIG. 1C revealed that it was a novel RING-H2finger protein, designated “Rbx1” or “Ring-box protein.”

Sequencing of Rbx1

The VHL tumor suppressor complex was fractionated by 13%SDS-polyacrylarnide gel electrophoresis. Proteins were visualized bystaining with Coomassie blue, excised, and subjected to in-gelreduction, S-carboxyamidomethylation, and tryptic digestion. Using 10%of the digestion mixture, peptide sequences were determined in a singlerun by microcapillary reversed-phase chromatography coupled to theelectrospray ionization source of a quadrupole ion trap massspectrometer (Finnigan LCQ). The ion trap's online data-dependent scansallowed the automatic acquisition of high resolution spectra todetermine charge state and exact mass, and tandem mass spectrometryspectra for sequence information. The relative collision energy was 35%AND ISOLATION WIDTH WAS 2.5 Dalton. Searches of the EST databaseperformed using TLASTIN algorithm identified human and mouse ESTs thatencoded the peptide sequences NHIMDLCIECQAN (SEQ ID NO:10),QVCPLDNREWEFQK (SEQ ID NO:11), WNAVAL (SEQ ID NO:12) and WLK which weredetermined by ion trap mass spectrometry of the 16 kDa polypeptide thatcopurified with the VHL complex. The identification was facilitated withthe algorithm SEQUEST (Eng, et al. 1994. J Am Soc Mass Spectrom 5:976)and by programs developed in the Harvard Microchemistry Facility(Chittum et al. 1998. Biochemistry 37:10866). I.M.A.G.E. Consortium CDNAcolonies (“I.M.A.G.E. Consortium: an integrated molecular analysis ofgenomes and their expression,” Genomics 33:151-152) encoding thecomplete 108 amino acid long ORFs of human (H71993) and mouse (W66989and AA260889) Rbx1 were obtained from Research Genetics, Huntsville,Ala., and the nucleotide sequences of both strands were determined.Human and mouse cDNAs encoded identical polypeptides of 108 amino acids.The amino acid sequence for human and mouse Rbx1 is shown in FIG. 2 andin SEQ ID NO:1. The nucleotide sequence for the human Rbx1 DNA is shownin nucleotides 7-333 or SEQ ID NO:3 and the nucleotide sequence for themurine Rbx1 DNA is shown in nucleotides 18-344 or SEQ ID NO:5, inclusiveof the stop codon. Nucleotides 1-6 and 1017 are 5′ untranslated regions,respectively and 334-508 and 345-504 are 3′ untranslated regions,respectively.

As shown in FIG. 2, Rbx1 is highly homologous to D. melanogaster ORF115C2.11, C. elegans ORF ZK287.5, and S. cerevisiae ORF YOL133w. Theamino acid sequence for Saccharomyces cerevisiae Rbx1 is shown in SEQ IDNO:2 and the nucleotide sequence for the Saccharomyces cerevisiae Rbx1DNA is shown in nucleotides 4-369 of SEQ ID NO:4, inclusive of the stopcodon. Comparison of the deduced amino acid sequences demonstrates thatthe proteins are highly homologous with about an 80 percent identity andthat the proteins are substantially the same. In addition, Rbx1 exhibitssignificant sequence similarity with Saccharomyces cerevisiaeAnaphase-Promoting Complex subunit APC11 (Zachariae, et al. 1998. “Massspectrometric analysis of the anaphase-promoting complex from yeast:Identification of a subunit related to cullins,” Science 279:1216-1219).

Because of the degeneracy of the genetic code, a DNA sequence may varyfrom that shown in SEQ ID NO:3 and still encode a Rbx1 protein havingthe amino acid sequence shown in SEQ ID NO:1. Such variant DNA sequencesmay result from silent mutations, e.g., occurring during PCRamplification, or may be the product of deliberate mutagenesis of anative sequence. The invention, therefore, provides equivalent isolatedDNA sequences encoding biologically active Rbx1 selected from: (a) thecoding region of a native Rbx1 gene; (b) cDNA comprising the nucleotidesequence presented in SEQ ID NO:3; (c) DNA capable of hybridization tothe native Rbx1 gene under moderately stringent conditions and whichencodes biologically active Rbx1; and (d) DNA which is degenerate as aresult of the genetic code to a DNA defined in (a), (b), or (c) andwhich encodes biologically active Rbx1. Rbx1 encoded by such DNAequivalent sequences are encompassed by the invention.

Recombinant Expression for Rbx1

Isolated and purified recombinant Rbx1 is provided according to thepresent invention by incorporating the corresponding DNA into expressionvectors and expressing the DNA in a suitable host cell to produce theprotein.

Expression Vectors

Recombinant expression vectors containing a nucleic acid sequenceencoding the protein can be prepared using well known techniques. Theexpression vectors include a DNA sequence operably linked to suitabletranscriptional or translational regulatory nucleotide sequences such asthose derived from mammalian, microbial, viral, or insect genes.Examples of regulatory sequences include transcriptional promoters,operators, enhancers, mRNA ribosomal binding sites, and appropriatesequences which control transcripion and translation initiation andtermination. Nucleotide sequences are “operably linked” when theregulatory sequence functionally relates to the DNA sequence for theappropriate protein. Thus, a promoter nucleotide sequence is operablylinked to a Rbx1 DNA sequence if the promoter nucleotide sequencecontrols the transcription of the appropriate DNA sequence.

The ability to replicate in the desired host cells, usually conferred byan origin of replication and a selection gene by which transformants areidentified, may additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with Rbx1 can be incorporated into expressionvectors. For example, a DNA sequence for a signal peptide (secretoryleader) may be fused in-frame to the protein sequence so that theprotein is initially translated as a fusion protein comprising thesignal peptide. A signal peptide that is functional in the intended hostcells enhances extracellular secretion of the appropriate polypeptide.The signal peptide may be cleaved from the polypeptide upon secretion ofprotein from the cell

Host Cells

Suitable host cells for expression of Rbx1 include prokaryotes, yeast,archae, and other eukaryotic cells. Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammalian cellularhosts are well known in the art, e.g., Pouwels et al. Cloning Vectors: ALaboratory Manual, Elsevier, N.Y. (1985). The vector may be a plasmidvector, a single or double-stranded phage vector, or a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors also may be and preferably are introduced intocells as packaged or encapsulated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems could also be employed to produce the protein using RNAs derivedfrom the present DNA constructs.

Prokaryotes useful as host cells in the present invention include grainnegative or gram positive organisms such as E. coli or Bacilli. In aprokaryotic host cell, a polypeptide may include a N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in theprokaryotic host cell. The N-termiinal Met may be cleaved from theexpressed recombinant Rbx1 polypeptide. Promoter sequences commonly usedfor recombinant prokaryotic host cell expression vectors includeβ-lactamase and the lactose promoter system.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. To construct an expression vector using pBR322, anappropriate promoter and a DNA sequence are inserted into the pBR322vector. Other commercially available vectors include, for example,pKK223-3 (Pharrmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (PromegaBiotec, Madison, Wis., USA), and the pET (Novagen, Madison, Wis., USA)and pRSET (Invitrogen Corporation, Carlsbad, Calif., USA) series ofvectors (Studier, F. W., J. Mol. Biol. 219: 37 (1991); Schoepfer, R.Gene 124: 83 (1993)).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include T7, (Rosenberg, A. H., Lade, B. N., Chui,D-S., Lin, S-W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56,125-135), β-lactamase (penicillinase), lactose promoter system (Chang etal., Nature 275:615, (1978); and Goeddel et al., Nature 281:544,(1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, (1980)), and tac promoter (Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, p. 412 (1982)).

Yeasts useful as host cells in the present invention include those fromthe genus Saccharomyces, Pichia, K. Actmomycetes and Kluyveromyces.Yeast vectors will often contain an origin of replication sequence froma 2 μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylafion, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, (1980)) or other glycolytic enzymes (Holland etal., Biochem. 17:4900, (1978)) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexolinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer etal., Gene, 107:285-195 (1991). Other suitable promoters and vectors foryeast and yeast transformation protocols are well known in the art.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc Natl Acad Sci USA,75:1929 (1978). The Hinnen protocol selects for Trp.sup.+transformantsin a selective medium, wherein the selective medium consists of 0.67%yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine,and 20 μg/ml uracil.

Mammalian or insect host cell culture systems well known in the artcould also be employed to express recombinant Rbx1, e.g., Baculovirussystems for production of heterologous proteins in insect cells (Luckowand Summers, Bio/Technology 6:47 (1988)) or Chinese hamster ovary (CHO)cells for mammalian expression may be used. Transcriptional andtranslational control sequences for mammalian host cell expressionvectors may be excised from viral genomes. Commonly used promotersequences and enhancer sequences are derived from Polyoma virus,Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNAsequences derived from the SV40 viral genome may be used to provideother genetic elements for expression of a structural gene sequence in amammalian host cell, e.g., SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication. Exemplary expression vectors for use in mammalian hostcells are well known in the art.

Rbx1 and other Ring box proteins may, when beneficial, be expressed as afusion protein that has the Ring box protein attached to a fusionsegment. The fusion segment often aids in protein purification, e.g., bypermitting the fusion protein to be isolated and purified by affinitychromatography. Fusion proteins can be produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of the protein. Preferred fusionsegments include, but are not limited to, glutathione-S-transferase,β-galactosidase, a poly-histidine segment capable of binding to adivalent metal ion, and maltose binding protein.

Expression and Recovery

According to the present invention, isolated and purified Rbx1 may beproduced by the recombinant expression systems described above. Themethod comprises culturing a host cell transformed with an expressionvector comprising a DNA sequence that encodes the protein underconditions sufficient to promote expression of the protein. The proteinis then recovered from culture medium or cell extracts, depending uponthe expression system employed. As is known to the skilled artisan,procedures for purifying a recombinant protein will vary according tosuch factors as the type of host cells employed and whether or not therecombinant protein is secreted into the culture medium. When expressionsystems that secrete the recombinant protein are employed, the culturemedium first may be concentrated. Following the concentration step, theconcentrate can be applied to a purification matrix such as a gelfiltration medium. Alternatively, an anion exchange resin can beemployed, e.g., a matrix or substrate having pendant diethylaminoethyl(DEAE) groups. The matrices can be acrylamide, agarose, dextran,cellulose, or other types commonly employed in protein purification.Also, a cation exchange step can be employed. Suitable cation exchangersinclude various insoluble matrices comprising sulfopropyl orcarboxymethyl groups. Further, one or more reversed-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media (e.g., silica gel having pendant methyl or other aliphaticgroups), ion exchange-HPLC (e.g., silica gel having pendant DEAE orsulfopropyl (SP) groups), or hydrophobic interaction-HPLC (e.g., silicagel having pendant phenyl, butyl, or other hydrophobic groups) can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, are well known in the artand can be employed to provide an isolated and purified recombinantprotein.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification, or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

In another aspect, the present invention provides a protein complexuseful for screening for potential therapeutic agents that wouldinterfere with or augment Rbx1-dependent stimulation of addition ofubiquitin or a ubiquitin-like protein to any substrate targeted formodification by SCF complexes. The complex contains a cofactor proteinand one or more proteins selected from the group consisting of a cullinprotein, a substrate recognition protein, and a linker protein.Additional components, e.g., ATP, may be added to the solution orcomposition: containing the complex to facilitate complex formation andutilization.

In a preferred embodiment, the complex is a ubiquitin ligase proteincomplex that contains cullin proteins; substrate recognition proteins;linker proteins; and cofactor proteins. The complex preferably has oneprotein from each group but can have more that one protein from eachgroup if required or preferred. Preferably, the complex is formed fromthe proteins but the solution or compositions containing the complex maycontain additional components if needed to facilitate complex formationand utilization. For example, solutions or compositions containing thecomplex can comprise various combinations of (1) Skp1, Elongin C, orother linker protein; (2) β-TRCP, Cdc4, Grr1, or other F-box substraterecognition protein or VHL or other Elongin C-Binding substraterecognition protein; (3) CUL1, Cdc53, CUL2, or other cullin protein; (4)Cdc34, UBC5C, or another E2 ubiquitin conjugating enzyme; (5)phosphorylated or other appropriately modified substrate; (6) E1ubiquitin activating enzyme; (7) ATP; and (8) ubiquitin, GST-ubiquitin,GST-Ubiquitin^(RA), ubiquitin derivatives, or a ubiquitin-like proteinsuch as SUMO, NEDD8, or Rub 1.

Preferably, the complex is a ubiquitin ligase protein complex thatcontains a cullin protein, a substrate recognition protein, a linkerprotein, and a cofactor protein. Most preferably, the complex is anisolated and purified ubiquitin ligase protein complex comprising suchproteins.

Generally, the cullin proteins of the present invention are proteinsselected from the group consisting of Cdc53, Cullin 1 (CUL1), Cullin 2(CUL2), Cullin 3 (CUL3), Cullin 4A (CUL4A), Cullin 4B (CUL4B), andCullin 5 (CUL5), preferably CUL1 or CUL2. The substrate recognitionproteins are selected from the group consisting of F-box proteins suchas β-TRCP (HOS), Cdc4, Grr1, and other members of this protein familyand VHL and other Elongin C binding proteins, preferably F-box proteinsor VHL. The linker proteins are selected from the group consisting ofSkp1 or Elongin C. Many such cullin, substrate recognition, and linkerproteins are known in the art and can be used in the present invention.The cofactor proteins are the Ring box proteins of the presentinvention, preferably Rbx 1.

FIG. 5 shows a diagram of SCF and VHL ubiquitin ligase complexesillustrating the ubiquitin ligases of the present invention. The cullinprotein is Cdc53/Cul1 in the SCF complex and CUL2 in the VHL complex,the substrate recognition protein is a F-box protein in the SCF complexand the VHL protein in the VHL complex, and the linker protein is Skp1in the SCF complex and Elongin C in the VHL complex. The VHL complexalso contains Elongin B. The cofactor protein Rbx1 binds to the otherproteins and activates the ubiquitin ligase. The SCF ubiquitin ligasetargets such substrate proteins as Sic1, cyclins, β-catenin, NF-κBinhibitors such as IκBα, and the like, and VHL ubiquitin ligase complextargets such substrate proteins as hypoxia-inducible factor 1 (HIF1) andhypoxia-inducible factor 2 (HIF2)

The present invention also provides methods for screening for potentialtherapeutic agents that would interfere with or augment Rbx1-dependentstimulation of addition of ubiquitin or a ubiquitin-like protein to anysubstrate including those targeted for modification by SCF ofVHL-containing complexes. The methods require forming a complex in vitrothat contains a cofactor protein and one or more proteins selected fromthe group consisting of a cullin protein, a substrate recognitionprotein, and a linker protein; adding a test compound to interact withthe complex; and determining if the complex remains intact or isdisrupted by the compound. If the complex is disrupted, the compound islikely to be a therapeutic agent useful for the treatment of thecorresponding disease, e.g., cancer or inflammatory disease. Forexample, a complex can be formed in vitro between a Cdc53 and Rbx1. Atest compound can be added to interact with the complex. An assay, e.g.,SDS-PAGE and immunoblotting or other known techniques, can be conductedto determine if the complex remains intact or is disrupted. SCFubiquitin ligases control the stability of proteins including but notlimited to cyclins and cyclin dependent kinase inhibitors, whichregulate the cell cycle, and IκB, which regulates inflammatoryprocesses. If the complex is disrupted, the compound is a likelycandidate for an anti-cancer agent or an anti-inflammatory drug.

Preferably, the complex used in the methods is the ubiquitin ligaseprotein complex described herein, most preferably an isolated andpurified ubiquitin ligase protein complex.

In similar assays, Rbx1 can be used to screen for agents which augmentor inhibit the activity of other Cullin-containing ubiquitin ligases andof the VHL complex controlling the conjugation of ubiquitin orubiquitin-like proteins to various sets of target proteins. Thehypoxia-inducible transcription factor HIF1α is a likely target forubiquitination by the VHL complex (Maxwell et al “The tumor suppressorprotein VHL targets hypoxi-inducible factors for oxygen-dependentproteolysis” Nature 399:271, 1999). Hypoxia-inducible transcriptionfactors regulate the expression of hypoxia inducible genes includingvascular endothelial growth factor, which controls normalvascularization as well as vascularization of tumors. Hence, compoundsthat disrupt or interfere with the function of the VHL complex arelikely candidates for anticancer drugs or for drugs that promotevascularization.

In another aspect, the present invention provides methods for diagnosingthe predisposition of a patient to certain carcinomas. The invention isbased upon the discovery that the absence of VHL-associated proteins,i.e., Ring box proteins such as Rbx1, from certain patient tissues orbody fluids indicates that the patient is predisposed to certaincarcinomas. The method comprises collecting a tissue or body fluidsample from a patient, analyzing the tissue or body fluid for thequantity of Ring box protein in the tissue, and predicting thepredisposition of the patient to certain carcinomas based upon theamount of Ring box protein in the tissue or body fluid. Specifically,determination of Rbx1 protein levels in certain tissues permits specificand early, preferably before metastases occur, detection of carcinomasin the patient. Carcinomas that can be diagnosed using the presentmethod include, but are not limited to, clear cell renal carcinoma.

In another aspect, the present invention provides methods for treatingany of several enumerated Ring box protein associated carcinomas oraugmenting metabolically deficient systems (e.g., fibronectindeposition) in humans and other animals. One method comprisesadministering a therapeutically effective amount of a compound thatenhances or augments in vivo the expression of the target Rbx1 gene andenhances the in vivo the expression of the Ring box protein to a patientdiagnosed as having a Ring box protein associated carcinoma or cellulardeficienciey and having been diagnosed as deficient in Ring box protein,preferably Rbx1. In a preferred embodiment, the compound is a nucleobasecontaining a sequence of the nucleic acid sequence encoding the Ring boxprotein. After administration, the nucleic acid sequence is activated toproduce the Ring box protein and increase the amount of the Ring boxprotein in the cell. Increasing the amount of Ring box protein increasesthe activity of VHL tumor suppressor activity, ubiquitination,fibronectin deposition, and similar activities. Another method comprisesadministering a therapeutically effective amount of a Ring box proteinto a patient diagnosed as having a Ring box protein associated carcinomaor cellular deficiency and having been diagnosed as deficient in Ringbox protein, preferably Rbx1. Carcinomas that can treated using thepresent method include, but are not limited to, clear cell renalcarcinoma.

In another aspect, the present invention provides methods for evaluatingthe effectiveness of a therapeutic treatment for Ring box associatedcarcinomas. The method comprises collecting a tissue or body fluidsample from a patient suffering from a Ring box associated carcinomraand having been subjected to a therapeutic treatment for such carcinoma,determining the amount of Ring box protein in the tissue or body fluidsample, and comparing the determined amount or Ring box protein to astandard indicative of normal Ring box protein levels. The standard canbe averages of Ring box protein levels for normal patients butpreferably is the level of Ring box protein for the patient beingtreated before the treatment began. Elevated levels of Ring box proteincompared to the standard indicates that the treatment is effective.

EXAMPLE 1 Use of Rbx1 with SCF^(Cdc4) in In Vitro Sic1 UbiquitinationAssay

Rbx1 can be used as a research tool to better understand various complexmechanisms of cell ubiquitination. To test directly the role of Rbx1 inSCF^(Cdc4) function, a standard in vitro Sic1 ubiquitination assay thatis dependent upon Sic1 phosphorylation and the E2 Cdc4 was employed.(Skowyra, et al. 1997. Cell 91:209). SCF^(Cdc4) components wereco-expressed in insect cells in the presence or absence of mammalian oryeast Rbx1, and complexes were immunopurified through either MYC-taggedCdc53(MYC-Cdc53) or FLAG-tagged Skp1 (FLAG-Skp1) subunits.Immunopurified SCF^(Cdc4) complexes were supplemented withphosphorylated Sic1, Cdc4, E1 ubiquitin activating enzyme, ATP, andGST-Ub^(RA) prior to analysis of Sic1 conjugates by immunoblotting.GST-Ub^(RA) forms polyubiquitinated products only poorly, so Sic1conjugates are integrated into a ladder of bands differing by ˜35 kd,the size of GST-Ub^(RA). Under the reaction conditions used, low butdetectable amounts of Sic1-GST-Ub^(RA) conjugates were produced by theSCF^(Cdc4) complex after a 60 minute reaction. In the presence of eitheryeast or mammalian Rbx1, the accumulation of Sic1-GST-Ub^(RA) conjugateswas dramaticaly increased after 20 minutes, and substantial amounts ofhigher molecular mass conjugates accumulated after 60 minutes. A largefraction of phosphorylated Sic1 was converted to Sic1-GST-Ub^(RA)conjugates when reactions included either the anti-Cdc53 oranti-FLAG-Skp1 immune complexes; e.g., >95% or Sic1 was converted toconjugates in reactions containing the anti-Skp1 immune complex. Incontrast, less than 5% of the substrate was conjugated in the absence ofRbx1 by either Cdc53 or Skp1 imnune complexes, despite the presence oflarger amounts of Cdc4 or Cdc53. To examine the extent of activation andthe concentration dependence of Rbx1 activation, SCF^(Cdc4) complexeswere purified from insect cells co-expressing varying levels of MYC-Rxb1and then assayed for Sic1 ubiquitin conjugating activity. In the absenceof Rbx1, low levels of conjugates were observed. Increasing quantitiesof Rbx1 led to increasing levels of ubiquitination, with the maximalextent of activation approaching 20-fold. This estimate represents alower limit of the extent to which Rbx1 can increase the rate ofaccumulation of Sic1-GST-Ub^(RA) conjugates, because a large fraction ofthe phosphorylated Sic1 substrate was depleted at the end of thereactions. Immunoblot analysis of these complexes revealed that thelevels of Cdc53, Cdc4, and Skp1 were constant throughout the Rbx1titration.

EXAMPLE 2 Use of Rbx1 with SCF^(β-TRCP) to Stimulate IκBα Ubiquitination

As another example of utilizing Rbx1 as a research tool, Rbx1 can beused to stimulate ubiquitination of phosphorylated IκB in the presenceof E1 ubiquitin-activating enzyme, the E2 ubiquitin conjugation enzymeUBC5C, ATP, GST-Ub^(RA), and SCF^(β-TRCP) (which contains the F-boxprotein β-TRCP). (Yaron, et al. 1998. “Identification of the receptorcomponent of the IκBα-ubiquitin ligase,” Nature 396:590-594; Winston, etal. 1999. “The SCF^(β-TRCP)-ubiquitin ligase complex associatesspecifically with phosphorylated destruction notifs in IκBα andβ-catenin and stimulates IκBα ubiquitination in vitro,” Genes Dev13:270-283). In a preferred embodiment, complex of Rbx1 and SCF^(β-TRCP)can be purified from lysates of Hi5 or Sf21 insect cells infected withbaculoviruses encoding SCF subunits and Rbx1. Complexes can besupplemented with phosphorylated IκB prepared as described by Winston(Winston, et al. 1999. Genes Dev 13:270-283), UBC5 C, E1ubiquitin-activating enzyme, ATP, and GST-Ub^(RA). After a period ofincubation, reaction products can be subject to SDS-PAGE and analyzed byWestern blotting using antibodies against IκBα. Conjugation ofGST-Ub^(RA) to IκBα can result in the formation of a ladder of IκBαconjugates differing in size by ˜35 kD, the size of GST-Ub^(RA).Confirmation that Rbx1 can be used as a research tool has beenpublished, e.g. Tan et al, Mol Cell 3:527 (1999).

EXAMPLE 3 Use of Rbx1 with the VHL Complex to Reconstitute HIF1αUbiquitination

As another example of utilizing Rbx1 as a research tool, Rbx1 was usedin assays for ubqitination of HIF1α (Maxwell et at “The tumor suppressorprotein VHL targets hypoxiinducible factors for oxygen-dependentproteolysis” Nature 399:271, 1999) in the presence of E1ubiquitin-activating enzyme, the E2 ubiquitin conjugation enzyme UBC5a,ATP, GST-Ub^(RA), and a complex consisting of VHL protein, Elongin B,Elongin C, CUL2, and Rbx1. In a preferred embodiment, the VHL-containingcomplex is purified from Sf21 insect cells infected with baculovirusesencoding subunits of the VHL complex. VHL complexes are supplementedwith lysates from insect cells infected with baculoviruses encoding HIF1or HIF2 and with UBC5a, E1 ubiquitin activating enzyme, ATP, andGST-Ub^(RA). After a period of incubation, reaction products aresubjected to SDS-PAGE and analyzed by Western blotting using antibodiesagainst HIF1α. Conjugation of GST-Ub^(RA) to HIF1α results in theformation of a ladder of HIF1α conjugates differing in size by ˜35 kD,the size of GST-Ub^(RA).

EXAMPLE 4 Use of Rbx1 to Stimulate Rub1 (NEDD8) Modification of CullinProteins

Rbx1 was used to stimulate modification of the cullin proteins Cdc53 andCUL2 with the ubiquitin-like protein Rub1 (also known as NEDD8) in thepresence of Rub1-activating enzyme Uba3/Ula1, Rub1-conjugating enzymeUbcl2, ATP, GST-Rub1, and a complex consisting of Rbx1 and either Cdc53or Cul2 (Kamura et al “The Rbx1 subunit of SCF and VHL E3 ubiquitinligase activates Rub1 modification of cullins Cdc53 and Cul2” Genes Dev.13:2928, 1999). In one embodiment, the Cdc53-Rbx1 complex is purifiedfrom Sf21 cells infected with baculoviruses encoding Cdc53 and Rbx1.Complexes are supplemented with Uba3/Ula1, Ubc12, ATP, and GST-Rub1.After a period of incubation, reaction products are subjected toSDS-PAGE and analyzed by Western blotting using antibodies thatrecognize Cdc53. Conjugation of GST-Rub1 to Cdc53 results indisappearance of ummodified Cdc53 and appearance of a new, more slowlymigrating band corresponding to the Cdc53-GST-Rub1 conjugate. In anotherembodiment, the CUL2-Rbx1 complex is purified from SE21 cells infectedwith baculoviruses encoding CUL2 and Rbx1. Complexes are supplementedwith Uba3/Ula1, Ubc12, ATP, and GST-Rub1. After a period of incubation,reaction products are subjected to SDS-PAGE and analyzed by Westernblotting using antibodies that recognze CUL2. Conjugation of GST-Rub1 toCUL2 results in disappearance of unmodified CUL2 and appearance of anew, more slowly migrating band corresponding to the Cdc53-GST-Rub1conjugate.

EXAMPLE 5 Reconstitution of the VHL Complex

Reconstitution of the VHL complex and its subassemblies in insect cellsand with bacterially expressed proteins in vitro shows that Rbx1 caninteract with VHL, CUL2, and the Elongin BC complex.

Rbx1 and VHL were subctoned into pBacPAK8 with N-terminal His tags andN-terminal myc and C-terminal FLAG tags, respectively. CUL1 wasintroduced in to the same vector with a C-terminal HA tag, and CUL2 wasintroduced into pBacPAK-His1 with N-terminal His and HA tags, andrecombinant baculoviruses were generated using the BacPAK baculovirusexpression system (Clontech). The baculovirus vectors encoding S.cerevisiae CDC53 (Willems, et al. 1996. “Cdc53 targets phosphorylated G1cyclins for degradation by the ubiquitin proteolytic pathway,” Cell86:453-463) and Elongins B and C have been described (Kanura, et al.1998. “The Elongin BC complex interacts with the conserved SOCS-boxmotif present in members of the SOCS, ras, WD-40 repeat, and ankyrinrepeat FAMILIES,” Genes Dev 12:3872-3881). Sf21 cells were cultured inSf-900 II SFM with 5% fetal calf serum at 27° C. and coinfected withvarious combinations of baculoviruses encoding myc-Rbx1, FLAG-VHL,HA-CUL2, HPC4-Elongin B, and HSV-Elongin C. At 60 hours after infection,Sf21 cells were collected and lysed by gentle vortexing in ice-coldbuffer containing 40 mM Hepes-NaOH, pH 7.9, 150 mM NaCl, 1 mM DTT, 0.5%(v/v) glycerol, 5 μg/ml leupeptin, 5 μg/ml antipain, 5 μg/ml pepstatinA, and 5 μg/ml aprotinin. Lysates were centrifuged at 10,000×g for 20minutes at 4° C. The supernatants were used for immnunoprecipitations.

For the expression and purification of recombinant proteins in E. coli,full-length mouse Rbx1 was expressed in pRSET B (Invitrogen) withN-terminal 6-histidine and myc epitope tags. Human VHL was expressed inpRSET B with N-terminal 6-histidine and C-terminal FLAG epitope tags.Purification of recombinant proteins from inclusion bodies andexpression constructs for Elongins B and C have been describedpreviously. (Kamura, et al. 1998. Genes Dev 12:3872-3881).

The procedures for inmnunoprecipitations and Western Blotting are asfollows. Anti-T7 and anti-HSV antibodies were from Novagen. Anti-HA(12CA5) and anti-c-myc (9E10) antibodies were from Boebringer-Mannheim.Anti-FLAG (M2) was from Eastman Kodak. Anti-Elongin C monoclonalantibody was from Transduction Laboratories. Anti-VHL monoclonalantibody (Ig32) was from Pharmingen. Anti-Sic1 (yN-19 and yC-19) andanti-Cdc53 (yC-17) antibodies were from Santa Cruz Biotechnology, Inc.Anti-Elongin B rabbit polyclonal antibodies were described previously.(Garrett, et al. 1995. “Positive regulation of general transcription smby a tailed ubiquitin homolog,” Proc Natl Acad Sci USA 92:7172-7176).Anti-HPC4 monoclonal antibody (Stearns, et al. 1988. “The interaction ofa Ca²⁺-dependent monoclonal antibody with the protein C activationpeptide region. Evidence for obligatory Ca²⁺binding to both antigen andantibody,” J Biol Chem 263:826-832) was obtained from a colleague.Western blots and imnmunoprecipitations of insect cell lysate andrefolded bacterially expressed proteins were performed as described.(Kamura, et al. 1998. Genes Dev 12:3872-3881).

As illustrated in FIG. 3A, all five components of the VHL complex couldbe coimmunoprecipitated together from Sf21 cell lysates with eitheranti-FLAG (Lane 5) or anti-myc (Land 9) antibodies. In addition, Rbx1,VHL, and the Elongin CB complex could be coimmunoprecipitated from Sf21cell lysates with either anti-FLAG (Lane 6) or anti-myc (Lane 10)antibodies in the absence of CUL2; and Rbx1, CUL2, and Elongin BCcomplex could be coimmuoprecipitated from Sf21 cell lysates withanti-myc antibody in the absence of VHL (Lane 11). As shown in FIGS. 3Band 3D, Rbx1 could also be coimmunoprecipitated with anti-myc antibodywith either VHL, CUL2, or the Elongin BC complex from lysates of Sf21cells coinfected with either myc-Rbx1 and FLAG-VHL, myc-Rbx1 andHA-CUL2, or myc-Rbx1, HPC4-Elongin B, and HSV-Elongin C. Consistent withthese results, as shown in FIG. 3C, Rbx1 could be coimmunoprecipitatedwith VHL-Elongin BC and Elongin BC subassemblies reconstituted in vitrowith bacterially expressed FLAG-VHL, HPC4-elongin B, and HSV-Elongin C.

CUL2 is a member of the multiprotein Cullin family (Kipreos, et al.1996. “Cul-1 is required for cell cycle exit in C. elegans andidentifies a novel gene family,” Cell 85:829-839), which also includesthe SCF component CUL1 and its S. cerevisiae homolog Cdc53. Because Rbx1interacted with CUL2 as shown in FIG. 3A, the possibility that it mightalso interact with CUL1 and Cdc53 was examined. As shown in FIG. 3D,Rbx1 binds both human CUL1 and S. cerevisiae Cdc53. Myc-Rbx1 and humanHA-CUL1 could be coimmunoprecipitated with anti-myc antibodies fromlysates of Sf21 cells coinfected with baculoviruses encoding myc-Rbx1and HA-CUL1. In addition, myc-Rbx1 and Cdc53 could becoimmunoprecipitated with anti-myc antibodies form lysates of Sf21 cellscoinfected with baculoviruses encoding myc-Rbx1 and Cdc53.

EXAMPLE 6 Effect of Rbx1 on Function of SCF^(Cdc4) Ubiquitin Ligase

Cdc53/CUL1 is a component of the recently described SCF ubiquitin ligasecomplex, which catalyzes ubiquitination of a diverse collection ofproteins with critical roles in cell cycle, transcription, anddevelopment. (Patton, et al. 1998. “Cdc53 is a scaffold protein formultiple Cdc34/Skp1/F-box protein complex that regulate cell divisionand methionine biosynthesis in yeast,” Genes Dev 12:692-705; Bai, et al.1996. “SKP1 connects cell cycle regulators to the ubiquitin proteolysismachinery through a novel motif, the F-box,” Cell 86:263-274; Patton, etal. 1998. “Combinatorial control in ubiquitin-dependent proteolysis:don't Skp the F-box hypothesis,” Trends Biochem Sci 14:236-243; Skowyra,et al. 1997. “F-box proteins are receptors that recruit phosphorylatedsubstrates to the SCF ubiquitin-ligase complex,” Cell 91:209-219; andFeldman, et al. 1997. “A complex of Cdc4p, Skp1p, and Cdc53p/Cullincatalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p,”Cell 91:221-230). SCF complexes include Cdc53/CUL1, Skp1, and one of thevariety of F-box proteins, which recruit substrates to the SCF forubiquitination. (Patton, et al. 1998. Genes Dev 12:692-705; and Bai, etal. 1996. Cell 86:263-274). The results demonstrate that Rbx1 canassemble into complexes containing CUL1 and the additional SCF componentSkp1, since Myc-Rbx1 could be co-iunmunoprecipitated with anti-T7antibodies from lysates of Sf21 cells coinfected with viruses encodingmyc-Rbx1, T7-tagged Skp1 (T7-Skp1), and HA-CUL1.

The WD40 repeat protein Cdc4, which recruits the cdk inhibitor Sic1 forubiquitination by the SCF^(Cdc4) complex, and the leucine-rich repeatprotein Grr1, which recruits G1 cyclin Cln2 for ubiquitination by theSCF^(Grr1) complex, are among several F-box proteins found in yeast.Having shown that Rbx1 interacts in cells not only with CUL2 and the VHLcomplex but also with the SCF components Cdc53/CUL1 (FIG. 3A) and Skp1,mutant yeast strains lacking the chromosomal RBX1 gene were generated inorder to test the possibility that Rbx1 might affect functions ofSCF^(Cdc4) and SCF^(Grr1).

A S. cerevisiae strain lacking the Rbx1 gene was constructed as follows.The Rbx1 gene was disrupted in MCY453 (Mata/MAThis3)-200/his3)-200can1R/can1R cyh2Rcyh2R ura3/ura3 leu2/leu2 trp1/trp1 lys2/lys2) byreplacing the complete Rbx1 ORF (YOL133w) with the HIS3 gene (Baudi etal. 1993. “A simple and efficient method for direct gene deletion inSaccharomyces cerevisiae. ” Nucleic Acids Res 21:3329-3330). For rescueof the rbx1 deletion strain (MCY557) with mammalian RBX1, wild type andmutant mammalian RBX1 genes were fused to the GAL1,10 promoter in theplasmid Yep352-GAL. These plasmids were transformed into MCY557 andURA3+ transformants were selected. Random spores were germinated ongalactose medium minus histidine and uracil and allowed to germinate for4 days at 30° C. The resulting colonies were tested for mating. Toconfirm that rescue was due to the presence of the SCF4 expressionplasmid, cells were tested for the ability to grow in FOA afterprolonged growth in medium containing uracil. Sporulation and tetraddissection showed 2:0 segregation for viability, and all viable sporeswere HIS⁻, indicating the RBX1 is an essential gene. Inviable sporesproduced ricrocolonies of 10-20 cells, many of which were abnormallyelongated or contained multiple, abnormally shaped buds. S. cerevisiaestrains containing mutations in genes encoding the SCF components Cdc53,Skp1, Cdc4, and Cdc34 exhibit a similar morphology. This phenotype wasdue to the RBX1 deletion, because the rbx1 deletion strain could berescued by expression of yRbx1.

The Rbx1 deletion strain was also rescued by expression of eithermyc-tagged mammalian Rbx1 (mRbx1 ) or a mutant mRbx1 (M4), in whichputative ring finger cysteine 53 and cysteine 56 were replaced withserines. The rbx1 deletion strain was not rescued, however, byexpression of a mutant mRbx1 (M3), in which putative ring fingercysteine 42 and cysteine 45 were replaced by serines. When expressed ineither the rbx1 deletion strain or in a wild type background, myc-taggedmRbx1 coimmunoprecipitated with the endogenous yeast Cdc53 protein,suggesting that it associates with Cdc53 in cells (FIG. 4A).Furthermore, coimmunopecipitation of wild type and mutant mRbx1 proteinswith Cdc53 correlated with their abilities to rescue the deletionphenotype, since significantly more Cdc53 was coimmunoprecipitated withthe complementating mRbx1 M4 mutant than with the noncomplementating M3mutant (FIG. 4A).

These results show that yeast Rbx1 (yRbx1 ) is a subunit and activatorof the SCF^(Cdc4) ubiquitin ligase and that mammalian Rbx1 (mRbx1 ) cansubstitute for its yeast counterpart in reconstitution of the activeSCF^(Cdc4) complex.

The SCF^(Cdc4) complex is required for ubiquitination and targeteddegradation of a variety of cell cycle regulatory proteins includingSic1, whose degradation is essential for the G1/S transition. TheSCF^(Grr1) complex is required for ubiquitination and targeteddegradation of the G1 cyclin Cln2. As one means of addressing the roleof Rbx1 in these processes, myc-tagged wild type or M4 mutant mRbx1 wereexpressed in the rbx1 deletion strain on a high copy number plasmidunder control of the GAL1,10 promoter. When cells carrying the plasmidwere shifted from galactose medium to glucose medium, Rbx1 protein wasdepleted, and the fraction of cells exhibiting the elongated budmorphology increased dramatically (FIG. 4C). Cells expressing the M4mutant arrested growth within a few hours of the glucose shift, whereascells expressing wild type Rbx1 continued to grow slowly, presumably dueto the presence of a small amount of residual Rbx1. As expected if M4cells arrest at least in part due to their inability to ubiquitinate thecell cycle regulators Sic1 and Cln2, M4 cells accumulated Sic1 and Cln2proteins when shifted into glucose (FIG. 4B).

Showing that Rbx1 is a general subunit of SCF ubiquitin-ligasesdemonstrates (1) that Rbx1 interacts with subunits of Cdc53/CUL1containing SCF ubiquitin-ligase complexes, (2) that the abilities ofRbx1 mutants to bind the Cdc53 subunit of SCF ubiquitin ligase correlatewith their abilities to prevent cell cycle arrest in a yeast strainlacking chromosomal Rbx1, and (3) that depletion of Rbx1 from yeastinterferes with the function of SCF^(Cdc4) and SCF^(Grr1). Having shownthat Rbx1 is also a component of the CUL2-containing VHL complex, Rbx1can now be applied in delineating the function of the VHL complex andperhaps other Cullin complexes as possible ubiquitin ligases andubiquitin-like ligases for as yet unidentified sets of target proteins.Since the multiprotein VHL complex has been shown to have roles in cellcycle regulation through its control of the levels of the cdk inhibitorp27, in repression of hypoxia-inducible genes, and in assembly of theextracellular fibronectin matrix, Rbx1 can be applied in thereconstitution of VHL complexes to determine therapeutics that cause thedissociation of the VHL complex. Further, Rbx1 is a component of theSCF^(Cdc4) complex, functioning as a common SCF subunit andparticipating in regulation of ubiquitination by SCF complexescontaining additional F-box proteins, including MET30 and β-TRCP, whichdirect ubiquitination of such target proteins as Swel and thetranscriptional regulators IκB and β-catenin. Thus, Rbx1 can be appliedin determining therapeutics capable of regulating ubiquitination viaother SCF complexes.

The present invention relates to a component in VHL tumor suppressoractivity found in the majority of sporadic clear cell renal carcinomasas well as autosomal dominant familial cancer syndrome that predisposesaffected individuals to a variety of tumors including clear cell renalcarcinomas, cerebellar hemangioblastomas and hemangiomas, retinalangiomata, and pheochromocytomas; general repression ofhypoxia-inducible genes; and regulation of p27 protein stability andfibronectin matrix assembly. The Ring box proteins described act as acellular marker useful (1) in detecting a possible predisposition tocertain carcinomas, (2) as molecular targets for treating thosecarcinomas therapeutically, (3) as a target for inhibition by drugs thatmanipulate the growth of cells, and (4) as a research tool to betterunderstand the various complex mechanisms of cell ubiquitination,binding of certain activator proteins, fibronectin deposition and otheraspects of the cellular division process.

While the preferred embodiments are shown to illustrate the invention,numerous changes to the materials and methods can be made by thoseskilled in the art. All such changes are encompassed within the spiritof the invention as defined by the appended claims.

1. An isolated and purified biologically active Ring box proteincomprising a polypeptide having an amino acid sequence corresponding toSEQ ID NO:1.
 2. A recombinant Ring box protein comprising a polypeptidehaving an amino acid sequence corresponding to SEQ ID NO:1.
 3. The Ringbox protein of claim 1, wherein the polypeptide is human.
 4. A Ring boxprotein according to claim 2, wherein the polypeptide is encoded by anucleic acid comprising the sequence of SEQ ID NO:3.
 5. A Ring boxprotein according to claim 2, prepared by the method of culturing anisolated cell containing an expression vector comprising a nucleic acidmolecule comprising the sequence of SEQ ID NO:3 under conditionssuitable for expression of the nucleic acid molecule.
 6. A proteincomplex comprising the Ring box protein of claim 1 or claim
 2. 7. Theprotein complex of claim 6, further comprising one or more proteinsselected from the group consisting of a cullin protein, a substraterecognition protein, and a linker protein.
 8. The protein complex ofclaim 7 wherein the complex is a ubiquitin ligase protein complex. 9.The protein complex of claim 8 wherein the ubiquitin ligase proteincomplex is selected from the group consisting of SCF and VHL.
 10. Theubiquitin ligase protein complex of claim 8 wherein the cullin proteinis selected from the group consisting of Cdc53, Cullin 1, Cullin 2,Cullin 3, Cullin 4A, Cullin 4B, and Cullin
 5. 11. The ubiquitin ligaseprotein complex of claim 8 wherein the substrate recognition protein isselected from the group consisting of β-TRCP, Cdc4, Grr1, VHL andElongin C binding proteins.
 12. The ubiquitin ligase protein complex ofclaim 8 wherein the linker protein is selected from the group consistingof Skp 1 and Elongin C.